BMDCs were generated from the femoral and tibial bone marrow cells of female mice as described previously49 . Cells were incubated in RPMI 1640 (Sigma-Aldrich, St Louis, MO) supplemented with 10% heat-inactivated fetal calf serum, 100 U/mL of penicillin, 100 μg/mL streptomycin, 100 μM 2-mercaptoethanol, 10 μM Minimum Essential Medium nonessential amino acid solution, and 20 ng/mL of murine GM-CSF (PeproTech, London, United Kingdom) for 10 days. CD11c+ cells were isolated by using the MACS separation system with anti-mouse CD11c MicroBeads (#130-052-001) and an autoMACS (all from Miltenyi Biotech, Tubingen, Germany). Splenic DCs were also isolated from mouse spleen by using the MACS separation system with anti-CD11c MicroBeas50 (link).
Mouse DC line JAWSII (CRL-11904, American Type Culture Collection, Manassas, VA) were maintained in MEM-α (GIBCO) supplemented with 20% FCS, 4 mM L-glutamine, 1 mM sodium pyruvate, 100 U/mL of penicillin, 100 μg/mL streptomycin, and 5 ng/mL of murine GM-CSF. All cells were incubated at 37 °C in a humidified atmosphere in the presence of 5% CO2.
LPS (#L3024) and poly I:C (#P0913) were purchased from Sigma-Aldrich. CpG-ODN (#tlrl-1826) was obtained from InvivoGen (San Diego, CA).
Mouse DC line JAWSII (CRL-11904, American Type Culture Collection, Manassas, VA) were maintained in MEM-α (GIBCO) supplemented with 20% FCS, 4 mM L-glutamine, 1 mM sodium pyruvate, 100 U/mL of penicillin, 100 μg/mL streptomycin, and 5 ng/mL of murine GM-CSF. All cells were incubated at 37 °C in a humidified atmosphere in the presence of 5% CO2.
LPS (#L3024) and poly I:C (#P0913) were purchased from Sigma-Aldrich. CpG-ODN (#tlrl-1826) was obtained from InvivoGen (San Diego, CA).
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